This Kirschstein-NRSA Postdoctoral Fellowship (F32) will provide George Dalton with research training under the guidance of Dr. Allyn Howlett in the Department of Physiology and Pharmacology at Wake Forest University Health Sciences that will allow him to develop into a successful, independent scientist. The long- term goal of the proposed research in this application is to characterize the CB1receptor (CB 1R)-mediated signaling events that regulate the tyrosine (Tyr) phosphorylation of proteins in neurons. Dr. Dalton has established that cannabinoid agonists activate CB1Rs to induce the Tyr phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) in N18TG2 (N18) neuroblastoma cells. These findings form the basis for the investigation of two specific aims in this proposal that will involve the elucidation of the mechanisms by which CB1Rs regulate the Tyr phosphorylation of FAK and ERK in N18 cells. Aim 1 will test the hypothesis that CB1Rs couple selectively to specific Ga i/o proteins to induce ERK and FAK Tyr phosphorylation in neurons. Aim 2 will test the hypothesis that CB1R-mediated ERK and FAK Tyr phosphorylation in neuronal cells involves (1) the transactivation of specific receptor tyrosine kinases (vascular endothelial growth factor receptor, epidermal growth factor receptor), (2) the activation of MAPK phosphatases, and (3) induction of neuronal differentiation of neuroprogenitor cells. To accomplish the studies outlined in this proposal, in vitro models of cell culture, specific kinase and phosphatase inhibitors, siRNA technology, and a novel high throughput In-Cell Western assay developed by LI-COR Biosciences will be utilized. In addition, N18 cells will be used that have been stably transfected with plasmids for Ga i1c351g, Gai2c352g, Gai3c351g, and Gao c351g that confer pertussis toxin resistance to the G1 subunit. Pertussis toxin will be used to acutely inactivate Ga i/o subtypes which will allow CB1Rs to couple to a single pertussis toxin-resistant G1 c351/352g i/o i/o subtype and the role that this specific Ga i/o protein plays in CB1R-mediated ERK/FAK activation can be determined. From a public health perspective, the endocannabinoid system is a target for pharmacotherapy for the treatment of pain, addictions, and neurodegenerative disorders. Protein kinases, protein phosphatases, and RTK transactivation play a central role in the endocannabinoid-mediated signaling networks that regulate cellular behavior and also represent potential drug targets in disease. These studies will contribute to the growing body of evidence that CB1R agonists and antagonists have therapeutic benefits in the treatment of substance abuse and neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: The endocannabinoid system is a target for the development of drugs that treat pain, addictions, and neurodegenerative disorders. Protein kinases, protein phosphatases, and GPCR-mediated RTK transactivation play a central role in the complex signaling networks that regulate cellular behavior and represent potential drug targets in disease. From a public health perspective, these studies will contribute to the growing body of evidence that CB1 receptor agonists and antagonists have therapeutic benefits in modulating cellular processes that involve synaptic plasticity and neuronal remodeling in pathologies such as substance abuse and neurodegenerative diseases.